Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction

Highlights

• Human bone tissue model, mimicking 3D-networked primary osteocytes

• Replicated the cell density, phenotype ofprimary human osteocytes in vitro

• Examined the model’s utility in replicatingmechanotransduction function and response to PTH treatment of osteocytes

Abstract

Osteocytes, residing as 3-dimensionally (3D) networked cells in bone, are well known to regulate bone and mineral homeostasis and have been recently implicated to interact with cancer cells to influence the progression of bone metastases. In this study, a bone tissue consisting of 3D-networked primary human osteocytes and MLO-A5 cells was constructed using: (1) the biomimetic close-packed assembly of 20–25 μm microbeads with primary cells isolated from human bone samples and MLO-A5 cells and (2) subsequent perfusion culture in a microfluidic device. With this 3D tissue construction approach, we replicated ex vivo, for the first time, the mechanotransduction function of human primary osteocytes and MLO-A5 cells by correlating the effects of cyclic compression on down-regulated SOST and DKK1 expressions. Also, as an example of using our ex vivo model to evaluate therapeutic agents, we confirmed previously reported findings that parathyroid hormone (PTH) decreases SOST and increases the ratio of RANKL and OPG. In comparison to other in vitro models, our ex vivo model: (1) replicates the cell density, phenotype, and functions of primary human osteocytes and MLO-A5 cells and (2) thus provides a clinically relevant means of studying bone diseases and metastases.

Introduction

Osteocytes reside as 3D-networked cells within mineralized extracellular matrix (ECM) cavities (“lacunae”) in bone tissue, and are interconnected by dendritic cell processes and gap junctions along ECM canals (“canaliculi”) [1], [2], [3], [4]. Osteocytes function as master regulators of homeostatic bone remodeling [1], [2], [3], and play important roles in the metabolic regulation of minerals [4]. Also, recent studies suggest that osteocytes, as 3D-networked cells, can interact with bone marrow cells [5] as well as prostate cancer and multiple myeloma cells located on the bone marrow side [6], [7], [8]. For bone homeostasis, osteocytes regulate: (1) osteoblastogenesis through releasing sclerostin and DKK1 and (2) osteoclastogenesis by secreting RANKL and OPG [1], [9], [10].

Our long-term motivation has been to construct the 3D-networked structure of human primary osteocytes, as a clinically relevant means of developing high-throughput in vitro bone tissue models. For clinical relevance, the use of human primary osteocytes is critically important since: (1) immortalizing human cells into cell lines by gene transfection perturbs the cells' gene expression profiles and cellular physiology [11], [12], [13] and (2) cell lines cannot capture the genotypic and phenotypic heterogeneity of primary cells [12]. Also, the ability of such ex vivo models to recapitulate the mechanotransduction function of osteocytes is critical as a physiological pathway of regulating bone formation.

It is well established that bones mechanically behave as “elastic sponges” [14]. When they are cyclically compressed during physical body movements, the interstitial fluid within the lacunocanalicular structure of bone squeezes in and out. As a result, flow-induced shear stresses are generated on osteocytes. Osteocytes are known to sense shear stresses through cell body and dendritic processes [15], [16]. Upon sensing mechanical stimuli, osteocytes reduce the production of sclerostin (encoded by SOST) and DKK1, which activate osteoblasts for new bone formation [17], [18], [19]. Especially, the SOST/sclerostin signaling pathway has received much attention as a unique drug target for treating osteoporosis [20] and tumor-induced osteolytic lesions [21].

In the past, this mechanotransduction function could not be equivocally replicated in vitro due to: (1) the relatively insufficient SOST and FGF23 expressions of osteocytic cell lines [22], [23], [24], [25], [26] and (2) the difficulty of maintaining the phenotype of primary osteocytes due to their osteoblastic dedifferentiation and proliferation during 2D culture [27], [28]. Also, state-of-the-art 3D bone tissue models, developed by other investigators [29], [30], [31], [32], cannot replicate physiologically relevant cell-to-cell distance and strong expressions of SOST and FGF23 as the key markers of mature osteocytes. Note that FGF23 is a hormone expressed by osteocytes to regulate phosphate homeostasis, and is gaining importance since it has been recently implicated to facilitate prostate cancer progression [33] and to be elevated in multiple myeloma patients [34].

We previously found that the 3D bone tissue structure consisting of 3D-networked osteocytes could be constructed via: (1) biomimetic assembly with microbeads with an osteocytic cell line (MLO-A5) and primary cells isolated from murine and human bones and (2) subsequent perfusion culture in a microfluidic device [28], [35], [36], [37]. These findings suggested that the 3D construction: (1) mimics the lacunocanalicular structure of human bone tissue, (2) mitigates the osteoblastic dedifferentiation and proliferation of primary osteocytes encountered in 2D culture, (3) promotes the significant gene expression of mature osteocytes (SOST and FGF23), and (4) therefore reproduces ex vivo the phenotype of terminally differentiated, non-proliferating 3D-networked osteocytes.

The major aims of this study were to: (1) examine our ex vivo 3D tissue model's ability to replicate the mechanotransduction function of primary human osteocytes and (2) demonstrate the model's utility by confirming the previously reported findings that continuous parathyroid hormone (PTH) treatment decreases SOST and increases FGF23.